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Role of B Lymphocytes in Cell-Mediated Immunity I

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Role of B Lymphocytes in Cell-Mediated Immunity I ROLE OF B LYMPHOCYTES IN CELL-MEDIATED IMMUNITY I. Requirement for T Cells or T-Cell Products for Antigen-Induced B-Cell Activation BY S. M. WAHL AND D. L. ROSENSTREICH Downloaded from http://rupress.org/jem/article-pdf/144/5/1175/1267614/1175.pdf by guest on 01 October 2021 (From the Humoral Immunity and Cellular Immunology Sections, Laboratory of Microbiology and Immunology, National Institute of Dental Research, National Institutes of Health, Bethesda, Maryland 20014) Bone marrow-derived (B) lymphocytes are antibody-forming cell precursors which when appropriately activated differentiate into antibody-secreting cells. Activation of B lymphocytes by mitogens and certain thymic-independent anti- gens is thought to occur directly without cooperation of other cells (I, 2). In contrast, the antibody response to most soluble protein antigens is defective in the absence of stimulated T cells (3-5). In addition to differentiation into antibody-secreting cells, B cells have also been shown to be capable of producing the biologically active mediators (lymphokines) associated with cell-mediated immune responses once thought to be the exclusive function of T cells. While B cells can be activated nonspecifically by B-cell mitogens and by triggering at their C3 and Fc surface receptors (6), investigations in our laboratory (6, 7) and in others (8) suggest that B cells cannot be triggered by soluble protein antigens to produce lymphokines. In view of the failure of B cells to differentiate into antibody-producing cells in the absence of T cells, we considered it likely that the defect in lymphokine production might be due to a requirement for T cells. In the present study, we have investigated the mechanism of antigen-specific lymphokine synthesis by B cells. We have found that this B-cell function requires T-cell help and that this help is mediated by a soluble factor produced by antigen-activated T cells. Materials and Methods Animals and Immunization. 400-500 g male, Hartley guinea pigs (Buckberg Lab Animals, Tomkins Cove, N. Y.) were immunized with dinitrophenylated ovalbumin (DNP14-OA) 1 in com- plete Freund's adjuvant (CFA) (7) or with 3 ~g tetanus toxoid (T.T) (Department of Public Health, Boston, Mass.) in CFA. Spleens and/or oil-induced peritoneal exudate cells were obtained from these animals 2-4 wk later. Nonimmunized guinea pigs (>500 g) were used as a source of glycogen- induced peritoneal macrophages for chemotaxis assays. Separation of T and B Lymphocytes. T and B lymphocytes were separated from single cell ~Abbreviations used in this paper: BCAF, B-cell-activating factor; CFA, complete Freund's adjuvant; CTX, chemotactic factor; DNP-OA, dinitrophenylated ovalbumin; E, rabbit erythro- cytes; [3H]TdR, tritiated thymidine; LPS, lipopolysaccharide; MNL, monocyte or macrophage; PELs, peritoneal exudate lymphocytes; POL, polymerized flagellin; T.T., tetanus toxoid. THE JOURNAL OF EXPERIMENTAL MEDICINE • VOLUME 144, 1976 1175 1176 ROLE OF B LYMPHOCYTES IN CELL-MEDIATED IMMUNITY suspensions of the spleens of immunized guinea pigs as previously described (6). Briefly, T lymphocytes were eluted off a nylon wool column and further purified by repassage over a second nylon column. B lymphocytes were isolated by removing on Ficoll-Hypaque gradients T cells which had rosetted with rabbit erythrocytes (E). Repeating this procedure with the nonrosetted cells resulted in a population of cells representing 10-25% of the total spleen cells which were >95% Ig positive (6) and responded to B, but not T, cell mitogens. Lymphocytes were suspended in RPMI 1640 media (NIH Media Unit, Bethesda, Md.) containing 2 mM glutamine, 100 IU/ml penicillin, and 100 pg/ml streptomycin, but without serum and cultured as reported (6). Proliferation Assay. Cultures of T and B cells were pulsed with 1 ~Ci/ml tritiated thymidine ([SH]TdR) (sp. act 6.0 Ci/mmol; Schwarz/Mann Div., Becton, Dickinson & Co., Orangeburg, N. Y.) 4 h before harvest. The cultures were harvested by means of a modified Skatron automated harvester (Flow Laboratories, Inc., Rockville, Md.) onto glass fiber filters. The filters were placed in scintillation vials with 5 ml scintillation fluid for determination of incorporated ['~H]TdR in a Downloaded from http://rupress.org/jem/article-pdf/144/5/1175/1267614/1175.pdf by guest on 01 October 2021 Beckman Scintillation counter (Beckman Instruments, Inc., Fullerton, Calif.). Data are expressed as mean counts per min per culture (1 × 10e cells) for triplicate cultures. Chemotaxis Assay. Cell-free supernates from these cultures were assayed for their ability to induce macrophage migration across polycarbonate (Nuclepore, Neuroprobe, Inc., Bethesda, Md.) filters with 5-pm pores. The chemotaxis assays were carried out in modified Boyden chambers (6). Triplicate filters were run for each sample and migration quantitated by counting 20 oil immer- sion fields per filter. Data are expressed as the mean number of cells per oil immersion field - 1 SE for the triplicate filters. Production of Supernates from T Lymphocytes. Peritoneal exudate cells were obtained 4 days after injection of sterile mineral oil (Drakeol 6-VR; Penreco Inc., Butler, Pro) into the peritoneal cavities of immunized guinea pigs. The harvested cells were pooled, washed several times, suspended in warm RPMI 1640 containing 10% fetal calf serum, and applied to prewashed glass bead columns (9). The loaded columns were incubated 45 min at 37°C, in 5% CO2, and the nonadherent cells eluted with 100 ml of warm medium. These nonadherent peritoneal exudate lymphocytes (PELs) which are enriched in antigen-reactive T cells (10) were washed several times in serum-free medium and resuspended at 1 × 106/ml with 2 ~g/ml antigen or medium only and incubated for 20 h unless otherwise indicated. The cultures were centrifuged and the cell-free supernates frozen (-2O°C) until used. These supernates were added to B-lymphocyte cultures (0.5 ml at 2 × 106/ml) in varying amounts and the total volume of each culture adjusted to 1 ml with serum-free RPMI 1640. Mitogens, Antigens, and Drugs. Antigens used in these experiments included DNP-OA and T.T. Lipopolysaccharide (LPS) from Escherichia coli 055:B5, (Difco Laboratories, Detroit, Mich.) and polymerized flagellin (POL) prepared from Salmonella adelaide (11) were utilized as B-cell mitogens. Cycloheximide and actinomycin D were obtained from Sigma Chemical Co., St. Louis, Mo. Results T-Lymphocyte Requirement for Antigen.Induced Lymphokine Production by B Cells. B cells obtained by the selective removal of E-rosette forming T cells from the spleens of DNP-OA-sensitized guinea pigs were incapable of respond- ing to the thymic-dependent antigen DNP-OA as measured by proliferation and production of a lymphokine chemotactic for macrophages (MNL CTX) (Table I). However, these same B cells proliferated and produced MNL CTX when stimu- lated with the B-cell mitogens, LPS and POL (Table I). Previous studies have shown that addition of macrophages does not enable B cells to respond to DNP- OA (7), however, a B-cell response could require T cells. In order to determine whether the B cells could be induced to respond to DNP-OA in the presence of T cells, the purified B lymphocytes were reconstituted with increasing numbers of DNP-OA-primed T cells. As seen in Fig. 1, B cells alone did not respond to DNP- OA, but when reconstituted with as few as 10% T cells a significant amount of mediator production became evident in the cultures. Further addition of T cells S. M. WAHL AND V. L. ROSENSTREICH 1177 TABLE I Proliferation and Lymphokine Production by B Cells in Response to Mitogens and Antigens Stimulant [3H]TdR incorpora- Chemotactic ac- tion (E/C) tivity None* 1.05 9 -+ 3 DNP-OA 1.1 3 _+ 1 LPS 3.5 69 -+ 4 POL 7.1 87 _+ 4 * 1 × 106 B lymphoeytes/ml serum-free medium were cultured with 0.1 ml medium only, 2 ~g/ml DNP-OA, 10 /~g/ml LPS, or POL for 48 h. Downloaded from http://rupress.org/jem/article-pdf/144/5/1175/1267614/1175.pdf by guest on 01 October 2021 The cell-flee supernates were then assayed for chemotactie activity (mean number of macrophages per oil immersion field _+ 1 SE of triplicate filters). $ Parallel cultures were pulsed with 1 ~tCi [3H]TdR 4 h before harvest at 48 h and assayed for incorporation of [3H]TdR. Ratio of mean counts per minute of experimental to control cultures (E/C) is shown. I DNP-OA ~ MEDIUM 60 5O u c oE 1£ % S CELLS 100 90 75 50 25 10 0 % T CELLS 0 10 25 50 75 90 100 FIG. 1. Production of MNL CTX by B cells in the presence of T cells. Chemotactic activity measured in 48-h supernates of cultures containing a total of 1 × 10e B and/or T lymphocytes in a vol of I ml medium. Increasing numbers of T cells were added to decreasing numbers of B cells and these cultures were stimulated with 2 ~g/ml DNP-OA or with medium only. with coincident decrease in total B cells resulted in little enhancement of MNL CTX. T cells alone did not respond to DNP-OA due to the absence of macro- phages in these preparations (Fig. 1) (7). Replacement of T Cells by a Cell-Free Supernate from Antigen-Activated T Cells. Although suggestive that in the presence of small numbers of T cells (10%), the B cells were producing MNL CTX, it could not be excluded that the T cells themselves were responsible for production of the MNL CTX found in these cultures. However, if the T cells were triggering B cells to undergo mediator synthesis, this might be accomplished by a T-cell product capable of interacting with B cells in the presence of antigen.
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